Product Manual
OxiSelect™ Catalase Activity Assay Kit,
Colorimetric
Catalog Number
STA- 341
96 assays
FOR RESEARCH USE ONLY
Not for use in diagnostic procedures
Introduction
Oxidative stress is a physiological condition where there is an imbalance between concentrations of
reactive oxygen species (ROS) and antioxidants. However, excessive ROS accumulation will lead to
cellular injury, such as damage to DNA, proteins, and lipid membranes. The cellular damage caused by
ROS has been implicated in the development of many disease states, such as cancer, diabetes,
cardiovascular disease, atherosclerosis, and neurodegenerative diseases. Under normal physiological
conditions, cellular ROS generation is counterbalanced by the action of cellular antioxidant enzymes
and other redox molecules. Because of their potential harmful effects, excessive ROS must be promptly
eliminated from the cells by this variety of antioxidant defense mechanisms.
Hydrogen peroxide is an ROS that is a toxic product of normal aerobic metabolism and pathogenic
ROS production involving oxidase and superoxide dismutase reactions. Hydrogen peroxide is
poisonous to eukaryotic cells and in high doses can initiate oxidation of DNA, lipids, and proteins,
which can lead to mutagenesis and cell death. Catalase is an antioxidant enzyme omnipresent in
mammalian and non-mammalian cells that destroys hydrogen peroxide by dismutation. Eukaryotic
catalases are heme enzymes found in the liver, kidney, and erythrocytes in high concentrations while
the lowest concentrations are in the connective tissues. The enzyme is concentrated in the peroxisome
subcellular organelles.
Cell Biolabs’ OxiSelect™ Catalase Activity Assay is a fast and reliable kit for the direct measurement
of catalase activity from cell lysate, plasma, serum, whole blood, and tissue homogenates. Each kit
provides sufficient reagents to perform up to 96 assays in a microtiter plate including blanks, catalase
standards and unknown samples. Direct spectrophotometric detection of catalase activity with
ultraviolet light can cause interference from proteins and other biological components. The assay
utilizes visible light (520 nm), which reduces sample interference. The kit is designed for use in single
plate microplate readers as well as readers with high-throughput capabilities. Please read this entire
manual prior to performing the assay.
Assay Principle
Cell Biolabs’ OxiSelect™ Catalase Activity Assay involves two reactions. The first reaction is the
catalase induced decomposition of hydrogen peroxide H2O2 into water and oxygen. The rate of
disintegration of hydrogen peroxide into water and oxygen is proportional to the concentration of
catalase (See Reaction 1 in Figure 1). A catalase-containing sample can be incubated in a known
amount of hydrogen peroxide. The reaction proceeds for exactly one minute, at which time the catalase
is quenched with sodium azide. The remaining hydrogen peroxide in the reaction mixture facilitates the
coupling reaction of DHBS and AAP in conjunction with an HRP catalyst (See Reaction 2 in Figure 1).
The quinoneimine dye coupling product is measured at 520nm, which correlates to the amount of
hydrogen peroxide remaining in the reaction mixture.
CATALASE
Reaction 1:
2 H2O2
2 H2O + O2
HRP
Reaction 2:
2 H2O2 (Left over) + DHBS + AAP
Figure 1: Catalase Assay Principle.
2
Quinoneimine Dye
Related Products
1. STA-305: OxiSelect™ Nitrotyrosine ELISA Kit
2. STA-310: OxiSelect™ Protein Carbonyl ELISA Kit
3. STA-312: OxiSelect™ Total Glutathione (GSSG/GSH) Assay Kit
4. STA-330: OxiSelect™ TBARS Assay Kit (MDA Quantitation)
5. STA-340: OxiSelect™ Superoxide Dismutase Activity Assay Kit
6. STA-342: OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence)
7. STA-347: OxiSelect™ In Vitro ROS/RNS Assay Kit (Green Fluorescence)
8. STA-345: OxiSelect™ ORAC Activity Assay
9. STA-346: OxiSelect™ HORAC Activity Assay Kit
10. STA-832: OxiSelect™ MDA Competitive ELISA Kit
11. STA-838: OxiSelect™ HNE Competitive ELISA Kit
Kit Components
1. Catalase Standard (Part No. 234101): One 50 µL amber tube of 600,000 Units/mL.
2. Hydrogen Peroxide (Part No. 234102): One 100 µL amber tube of an 8.82 M solution.
3. Chromogenic Reagent A (Part No. 234103): One 0.5 mL amber tube.
4. Chromogenic Reagent B (Part No. 234104): One 30 mL bottle.
5. HRP Catalyzer (Part No. 234105): One 50 µL amber tube.
6. Assay Diluent (10X) (Part No. 234106): One 20 mL bottle.
7. Sample Buffer (5X) (Part No. 234107): One 50 mL bottle.
8. Catalase Quencher (Part No. 234108): One 10 mL amber bottle.
Materials Not Supplied
1. Sample extracts for testing
2. Standard 96-well microtiter plates for use in microplate reader
3. Distilled or deionized water
4.
5.
6.
7.
8.
9.
Bottles, flasks, and conical or microtubes necessary for reagent preparation
Reagents and materials necessary for sample extraction and purification
10 µL to 1000 µL adjustable single channel micropipettes with disposable tips
50 µL to 300 µL adjustable multichannel micropipette with disposable tips
Multichannel micropipette reservoir
Microplate reader capable of reading at 520 nm.
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Warnings and Precautions
• Hydrogen peroxide is corrosive and is harmful by inhalation or if swallowed. Contact with skin
may cause burns. In case of contact with eyes, rinse immediately with plenty of water and seek
medical attention. Liquid may develop pressure. Keep away from combustible materials.
• Sodium Azide is harmful in contact with skin or if swallowed. Contact with acids liberates a very
toxic gas. Azide may react with copper plumbing to form explosive azides. Flush with plenty of
water when pouring down a drain.
• Chromogenic Solutions may cause eye or skin irritation. May be harmful if swallowed. May cause
respiratory and/or digestive tract irritation.
Storage
Upon receipt, store the Catalase Standard, Hydrogen Peroxide, Chromogenic Solutions A and B, and
the HRP Catalyzer at 4°C. Store all remaining kit components at room temperature.
Preparation of Reagents
Note: All reagents must be brought to room temperature prior to use.
•
1X Assay Diluent: Dilute the 10X Assay Diluent stock to a 1X solution with distilled water. Mix
to homogeneity. Store the 1X Assay Diluent at room temperature.
•
1X Sample Buffer: Dilute the 5X Sample Buffer stock to a 1X solution with distilled water. Mix to
homogeneity. Use this for all sample and standard dilutions. Store the 1X Sample Buffer at room
temperature.
•
Hydrogen Peroxide Working Solution: Prepare a 12 mM Hydrogen Peroxide Working Solution by
diluting the stock 8.82 M H2O2 solution in Assay Diluent (eg. Add 5 µL of H2O2 stock to 3.67 mL
Assay Diluent). Prepare only enough Hydrogen Peroxide Working Solution necessary for
immediate applications. This reagent is stable for 2 weeks when stored at 2-8ºC and protected from
light.
•
Chromogenic Working Solution: Prepare only enough Chromogenic Working Solution necessary
for immediate applications. Prepare a Chromogenic Working Solution by diluting Chromogenic
Reagent A 1:100 with Chromogenic Reagent B. (Example: Add 0.10 mL of Chromogenic Reagent
A to 9.9 mL of Chromogenic Reagent B) Mix thoroughly. Next, add 1 µL of HRP Catalyzer per 1
mL of Chromogenic Working Solution. (Example: Add 10 µL to 10 mL of Chromogenic Working
Solution) Mix to homogeneity. This reagent is stable for 2 weeks when stored at 4ºC and protected
from light.
Preparation of Samples
Note: Samples should be stored at -70°C prior to performing the assay. Sample should be prepared at
the discretion of the user. The following recommendations are only guidelines and may be altered to
optimize or complement the user’s experimental design. Samples should be diluted in Sample Buffer
unless noted otherwise. Bovine serum albumin can be added to samples with a protein concentration <
0.050 mg/mL to stabilize the enzyme.
•
Whole Blood: Collect whole blood (WB) in an anticoagulant tube and mix by inversion.
Freeze down 100 µL of whole blood to lyse. Dilute the sample 1:1000 prior to use and use
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within 1 hour after making diluted preparations. Store any whole blood sample not being
immediately used at -70°C. Samples can be stored short-term at 4°C for 10 days, but must be
freeze-thawed to lyse cells.
•
Plasma: Collect blood with an anticoagulant such as heparin or citrate and mix by inversion.
Centrifuge a minimum of 0.5 mL whole blood at 2500 x g at 4°C for 5 minutes. Collect plasma
supernatant without disturbing the white buffy layer. Sample should be immediately used or
frozen at -70°C for storage. Using a centrifugal ultrafiltration tube with a 30kDa molecular
weight cut-off, tare the filter on an analytical balance. Add 125 µL of plasma to the filter tube.
Record the weight. Add 375 µL of Assay Diluent. Centrifuge at 10,000 x g for 30 minutes at
room temperature. Discard the filtrate. Reconstitute the retained fluid with Assay Diluent to the
original weight recorded. Mix solution thoroughly.
•
Erythrocyte Lysate: Collect blood in an anticoagulant tube and mix by inversion. Centrifuge a
minimum of 0.5 mL whole blood at 2500 x g at 4°C for 5 minutes. Discard the plasma
supernatant and wash 5 times in cold 0.9% NaCl. Resuspend the erythrocyte pellet with 4 x the
cell-packed volume with ice-cold deionized water. Incubate on ice for 10 minutes. Dilute the
sample 1:400 before use. Sample should be tested within 1 hour after making diluted
preparations. Centrifuge Sonicate or homogenize tissue sample on cold PBS and centrifuge at
10,000 x g for 10 minutes at 4°C. Aliquot and store the supernatant for use in the assay. Store
any unused lysate at -70°C for up to one month. Samples can be stored short-term at 4°C for 5
days.
•
Blood Serum: Collect blood in a tube with no anticoagulant. Allow the blood to clot at room
temperature for 30 minutes. Centrifuge at 2500 x g for 20 minutes. Remove the yellow serum
supernatant without disturbing the white buffy layer. Samples should be tested immediately or
stored at -70°C for up to one month.
•
Tissue Homogenate: Prior to dissection, perfuse tissue or rinse with a phosphate buffered saline
(PBS) solution. This is to remove any red blood cells and clots. Weigh and homogenize the
tissue on ice in 5-10 mL cold PBS with 1mM EDTA per gram of tissue. Centrifuge at 10,000 x
g for 15 minutes at 4°C. Remove the supernatant and store on ice. Store any unused
supernatant at -70°C for up to one month.
•
Cell Lysate: Collect cells by centrifuging at 2000 x g for 10 minutes at 4°C. For adherent cells,
do not use proteolytic enzymes to harvest cells, but rather use a rubber policeman. Sonicate or
homogenize the cell pellet on ice in 1-2 mL cold PBS, 1mM EDTA. Centrifuge at 10,000 x g
for 15 minutes at 4°C. Remove the supernatant and store on ice. Aliquot and store the
supernatant for use in the assay. Store any unused supernatant at -70°C for up to one month.
Preparation of Catalase Standard Curve
1. Use only enough Catalase Standard as necessary for immediate applications. Immediately prior to
use, vigorously vortex the Catalase Standard stock vial to obtain a homogenous suspension and
immediately remove the desired amount. It is recommended to pipette up and down several times
prior to removing the suspension. Prepare fresh standards by diluting the 600,000 Units/mL
Catalase Standard stock 1:60 in Sample Buffer for a 10,000 Units/mL solution. (Example: Add 5
μL of Catalase Standard stock tube to 295 μL of Sample Buffer)
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Note: One unit of catalase is the amount of enzyme that will decompose 1.0 µmole of H2O2 per
minute at 25°C.
2. Use the 10,000 Units/mL solution to prepare a series of catalase standards according to Table 1
below.
Note: If crystals are visible upon preparation, gently warm the standards at 30ºC with vortexing.
Tubes
1
2
3
4
5
6
7
8
10,000 Units/mL
Catalase Standard
Dilution (µL)
10
500 of Tube #1
500 of Tube #2
500 of Tube #3
500 of Tube #4
500 of Tube #5
500 of Tube #6
0
Sample Buffer (µL)
990
500
500
500
500
500
500
500
Catalase Concentration
(Units/mL)
100
50
25
12.5
6.25
3.125
1.5625
0
Table 1. Preparation of Standards
Note: Catalase is unstable at high dilutions and should be kept on ice while being used. Use diluted
standards within 60 minutes of preparation. Do not store diluted Catalase Standard solutions.
Assay Protocol
Note: Each catalase standard and samples should be assayed in duplicate or triplicate. A freshly
prepared standard curve should be used each time the assay is performed.
1. Add 20 µL of the diluted catalase standards or unknown samples to a 96-well microtiter plate.
2. Add 50 µL of the Hydrogen Peroxide Working Solution (12mM) to each well. Mix thoroughly and
incubate exactly 1 minute.
3. Stop the reaction by adding 50 µL of the Catalase Quencher into each well and mix thoroughly.
4. Transfer 5 µL of each reaction well to a fresh well.
5. Add 250 µL of the Chromogenic Working Solution to each well. Incubate the plate 40-60 minutes
with vigorous mixing.
6. Read the plate absorbance at 520 nm. Save values for Calculation of Results below.
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Example of Results
The following figure demonstrates a typical standard curve for the OxiSelect™ Catalase Activity
Assay. One should use the data below for reference only. This data should not be used to interpret or
calculate actual sample results.
1.8
1.6
1.4
OD 540nm
1.2
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
Catalase (U/mL)
Figure 2: Catalase Activity Assay Standard Curve.
Calculation of Results
The concentration of the unknowns can be determined by interpolation of the catalase standard curve
(Figure 2) or analyzed by plate reader software with a 4 parameter logistic curve fitting program to
calculate a second order polynomial regression for each sample.
A520 = ax2 + bx + c
The “A520” is the absorbance of the sample, “x” is the catalase activity in Units/mL, and a, b, and c are
the coefficients of the quadratic equation.
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To use Microsoft Excel,
1. Plot the catalase standard curve, similar as Figure 3, use OD 520 nm for X axis and Catalase
amount as Y axis.
2. Highlight the curve first. Select ‘trendline of a second order polynomial and display equation
on chart’.
3. Calculate the catalase activity using the chart equation.
120
Catalase ( U/mL)
100
y = 49.255x2 - 147.14x + 112.32
R2 = 0.998
80
60
40
20
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
OD 540nm
Figure 3: Catalase Activity Assay Standard Curve with second order polynomial equation.
Assay Compatibility
Since blood is one of the sources for catalase, anticoagulants such as heparin, potassium EDTA, or
sodium citrate have been tested for compatibility. Various endogenous compounds present in plasma
and serum, such as Ascorbic acid and Uric acid, may interfere with the assay. The serum/plasma
ascorbic acid and uric acid interference can be minimized by centrifugation with an ultrafiltration
device. Whole blood, RBC lysates, or other lysate/tissue sample levels should not cause interference
with the results.
Samples are usually diluted significantly before testing; however tissue samples with low catalase
concentrations may lead to a small dilution factor, which can cause endogenous compounds such as
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hemoglobin or albumin to interfere with the assay. Please see Table 2 below. Use the chart as a guide
for preparing samples prior to performing the assay.
Interfering Substance
Ascorbic acid
Albumin
Sodium citrate
Tripotassium EDTA
Hemoglobin
Heparin
Glucose
Triton X-100
Compatibility
10-20 µM
50 mg/mL
20 mM
4mM
0.75 mg/mL
14 Units/mL
5 mM
0.5%
Table 2: Assay Compatibility
References
1.
2.
3.
4.
5.
Ames, B.N., Shigenaga, M.K., and Hagen, T.M. (1993) Proc. Natl. Acad. Sci. USA 90: 7915-7922.
Aebi, H., (1984) Methods Enzymol. 105: 121-126.
Deisseroth, A., and Dounce, A.L. (1970) Physiol. Rev. 50: 319-375.
Fossati, P. et al. (1980) Clin. Chem. 26: 227-231.
Zamocky, M., and Koller, F. (1999) Prog. Biophys. Mol. Biol. 72: 19-66.
Recent Product Citations
1. Albalawi, A. et al. (2017). Protective effect of carnosic acid against acrylamide-induced toxicity in
RPE cells. Food Chem Toxicol. pii: S0278-6915(17)30034-0. doi: 10.1016/j.fct.2017.01.026
2. Parra, O. et al. (2016). Biochemical precursor effects on the fatty acid production in cell suspension
cultures of Theobroma cacao L. Plant Physiol. Biochem. 111:59-66.
3. Rababa’h, A. M. et al. (2016). Exposure to waterpipe smoke induces renal functional and oxidative
biomarkers variations in mice. Inhal Toxicol. doi:10.1080/08958378.2016.1210703.
4. Helgesen, K. O. et al. (2016). Increased catalase activity—A possible resistance mechanism in
hydrogen peroxide resistant salmon lice (Lepeophtheirus salmonis). Aquaculture.
doi:10.1016/j.aquaculture.2016.10.012.
5. Kurade, M.B. et al. (2016). Insights into microalgae mediated biodegradation of diazinon by
Chlorella vulgaris: Microalgal tolerance to xenobiotic pollutants and metabolism. Algal Res.
20:126-134.
6. Piemontese, M. et al. (2016). Low bone mass and changes in the osteocyte network in mice lacking
autophagy in the osteoblast lineage. Sci Rep. doi:10.1038/srep24262.
7. Ahmad, I. M. et al. (2016). Redox status in workers occupationally exposed to long-term low levels
of ionizing radiation: A pilot study. Redox Rep. Doi:10.1080/13510002.2015.1101891.
8. Erbaş, O. et al. (2016). Levetiracetam attenuates rotenone-induced toxicity: A rat model of
Parkinson's disease. Environ Toxicol Pharmacol. 42:226-230.
9. Harisa, G. I. et al. (2015). Influence of pravastatin chitosan nanoparticles on erythrocytes
cholesterol and redox homeostasis: An in vitro study. Arab J Chem.
doi:10.1016/j.arabjc.2015.10.016.
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10. Lee, Y. Y. et al. (2015). Exercise suppresses COX-2 pro-inflammatory pathway in vestibular
migraine. Brain Res Bull. 116:98-105.
11. Guven, M. et al. (2015). The neuroprotective effect of kefir on spinal cord ischemia/reperfusion
injury in rats. J Korean Neurosurg Soc. 57:335-341.
12. Cheng, Y. Y. et al. (2015). SIRT1-related inhibition of pro-inflammatory responses and oxidative
stress are involved in the mechanism of nonspecific low back pain relief after exercise through
modulation of Toll-like receptor 4. J Biochem. doi: 10.1093/jb/mvv041.
13. Timmusk, Salme. (2015). Sfp-type PPTase inactivation promotes bacterial biofilm formation and
ability to enhance wheat drought tolerance. Front Microbiol. 6:387.
14. Yener, A. U. et al. (2015). Effects of kefir on ischemia-reperfusion injury. Eur Rev Med Pharmacol
Sci. 19:887-896.
15. Torres, F. et al. (2015). Melatonin reduces oxidative stress and improves vascular function in
pulmonary hypertensive newborn sheep. J Pineal Res. doi: 10.1111/jpi.12222.
16. Plestina-Borjan, I. et al. (2015). Association of age-related macular degeneration with erythrocyte
antioxidant enzymes activity and serum total antioxidant status. Oxid Med Cell
Longev. 2015:804054.
17. Javanbakht, M. H. et al. (2015). Evaluation of antioxidant enzyme activity and antioxidant capacity
in patients with newly diagnosed pemphigus vulgaris. Clin Exp Dermatol. doi: 10.1111/ced.12489.
18. Mora, M. et al. (2015). Minocycline increases the activity of superoxide dismutase and reduces the
concentration of nitric oxide, hydrogen peroxide and mitochondrial malondialdehyde in manganese
treated Drosophila melanogaster. Neurochem Res. 39:1270-1278.
19. Markiewicz-Górka, I. et al. (2015). Effects of chronic exposure to lead, cadmium, and manganese
mixtures on oxidative stress in rat liver and heart. Arch Ind Hyg Toxicol. 66.
20. Girgih, A. T. et al. (2014). A novel hemp seed meal protein hydrolysate reduces oxidative stress
factors in spontaneously hypertensive rats. Nutrients. 6:5652-5666.
Please see the complete list of product citations: http://www.cellbiolabs.com/catalase-assay.
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